Research on synaptic vesicles (SVs) has been dominated for many years by studies that have focused on mechanisms for their exocytosis and endocytosis. By comparison, relatively little has been learned about the subject of this competitive renewal application, the trafficking of SVs in the axon terminal. The proposal is focused on two morphologically and functionally distinct populations of SVs, the readily releasable and reserve pools, and on the role of actin in SV recycling. The planned experiments are based on three technical advances that were made during the present funding period. 1) Several years ago, Dr. Betz pioneered the use of fluorescent FM dyes as tools for labeling SVs in axon terminals. Much more recently, he developed a method for converting DAB into an insoluble, electron dense product following photoactivation of FM dyes. This method leads to very high resolution labeling of individual SVs and putative SV recycling cisternae at the EM level. 2) An EM method for discriminating readily releasable SVs from the reserve pool of SVs has been developed. The method relies on FM dye photoactivation, and the distinct properties of two such dyes, FM1-43 and FM2-10. Apparently, FM1-43 labels both SV pools, whereas the less hydrophobic FM2-10 selectively labels the readily releasable population. 3) Two pools of actin are located in motor neuron synapses: beta-actin is confined to axon terminals, whereas alpha-actin is restricted to skeletal muscle fibers. Using an antibody specific for beta-actin, Dr. Betz has been able to achieve selective immunofluorescent labeling of the actin present in axon terminals, without interference from the nearby, intensely actin-rich muscle fibers. Using this approach, he has found that in resting axon terminals actin is localized in a ladder-like pattern which is complementary to where clusters of SVs are localized, but following prolonged nerve stimulation the actin adopts a more diffuse distribution. Dr. Betz interprets these results to indicate that actin is critically invoved in SV trafficking.